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Offlineshopdropper
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Amanita virosa]
    #19100163 - 11/07/13 12:08 PM (10 years, 3 months ago)

nice job. those brown colonies are the ones you want to use for innoculatin LC. further generations it will start to form the white dormant colonies. especially on PDA


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Amanita virosa]
    #19100839 - 11/07/13 02:18 PM (10 years, 3 months ago)

Quote:

Amanita virosa said:
Does it grow quickly?  If it does, it is often difficult  to detect contams even microscopically, as the myc over-runs them.  They then show up after transfer to grain.  But hopefully not, in your case.




Yeah, it grows quickly.  No latency, the honeydew starts growing within 12 hours.  I've been wondering what it really does with the contamination.  Whether it eats it or not, and if it does eat it then what happens?  Actually I suspect that the slightly whitish cast in the lower part of the plate in picture 1 may actually be contam, but of this I am not sure.  I won't be surprised if endemic contams show up at a later stage of growth, but I'm keeping my fingers crossed that they won't.

This also touches on Tana's question as to why I did not culture directly from the sclerotia to begin with.  Germinating the sclerotia and taking spore prints from the ascocarps should yield cultures that are completely clean, whereas isolation from sclerotium might still carry contamination.


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OfflineShroomDoom
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19100943 - 11/07/13 02:34 PM (10 years, 3 months ago)

:popcorn:


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19101386 - 11/07/13 03:44 PM (10 years, 3 months ago)

Quote:

shopdropper said:
nice job. those brown colonies are the ones you want to use for innoculatin LC. further generations it will start to form the white dormant colonies. especially on PDA




Thanks!  Does the brown pigment come from presence of LADs?  There is also orange, yellow, green and purple colors present in the mycelium and/or in the media.  Any idea what these colors indicate?

What do you think of the young mycelium glowing violet under black light?  Is that the color I'm looking for?


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19102639 - 11/07/13 08:12 PM (10 years, 3 months ago)

OK, Everybody!  I'm checking out of the Shroomery for now.  I'll come back to this if and when my Paspali sclerotia germinate.  Might be two weeks to a month from now.  I'll post photos of the germinated sclerotia then.  Thanks to everyone who posted here for their interest and for sharing their knowledge and experience.

- Mr.Caterpillar


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Offlineshopdropper
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19104506 - 11/08/13 09:21 AM (10 years, 3 months ago)

i dont think the (brown) color is coming from the alkaloids. yes the fluorescence is what your looking for that is a positive indication of alkaloids! im not sure of the other colors. I have heard of a violet colored mycelium it produces better than the white but not as good the brown colonies.



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DON'T TRY THIS AT HOME: no guarantees can be made about the accuracy of the information herein. The information dicussed in these posts is purely hypothetical, and for intelectual purposes only. Any similarity between internet chat and real life is pure coincidence.

:google:


Edited by shopdropper (11/28/13 06:28 PM)


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19221427 - 12/03/13 10:21 AM (10 years, 2 months ago)

It seems that the sclerotia never germinated.  I followed instructions as described in the old journal article cited above pretty carefully, but it didn't work.  The vials containing sclerotia were cold incubated at ~ 7 c for 1 month and then were kept at ~ 23 c for a month.  It seems to me that some of them (at least) should have germinated, but I see no sign of it.  Perhaps the way I cleaned the sclerotia was not correct, or somehow inadequate.

Considering how easy it was to obtain culture from the honeydew, on a purely pragmatic level I am not sure why one would want to bother with germinating the sclerotia, however I would like to see the little stroma forming, and it would be interesting to me to isolate culture from the stroma tissue.

I'm thinking of trying it again using "magic water crystals" as a germination substrate.  I didn't particularly like using sand as the article suggested and couldn't fathom why one would unless just following instructions that have been handed down.  And what would be wrong with germinating them in a glass with a rolled moist paper towel on the inside?  I think I can dig up some other articles with techniques of germinating ergot sclerotia, but haven't gotten around to it yet.

Anyone have any suggestions?

- Mr.Caterpillar


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19221604 - 12/03/13 11:12 AM (10 years, 2 months ago)

Fluorescence that I reported earlier was not the real McCoy.  That was just the white mycelium glowing as white stuff glows in UV-A.  However, since that time I have seen a lot of genuine glowing in the cultures.  Glow comes in three colors: Pink, Yellow and Orange.  I didn't see much glow until I put some plates in the fridge.  These plates turned a deep pink/magenta color* (I would not call it "violet"** - although I think that is what is meant in the literature when "violet" is referred to). The pink tissue fluoresces to hot pink under black light. 

Additionally, metabolic excretions in the agar can be seen to fluoresce. The glow on the underside of the plate is most intense along the outer growth parameter and is typically a yellow color.  Some cultures excrete yellow metabolites that fluoresce to an orange hue and the entire bottom of the plate will glow in that case.

With age these cultures turn various deep shades of ochre, orange, brown and purple.  When the mycelium and the media are heavily pigmented they do not glow, so glowing is more common in immature cultures.

Now I have cultures growing out on fluorescein dyed plates.  Considering that UV glow is the most practical indicator of ergoline alkaloids, I have selected tissue that glows.  In other words I have selected the purple mycelium that exudes concentrated yellow metabolites.  On the fluorescein plates these selections are growing out uniformally purple.  This was done only a couple of days ago, and I'll see later if the purple tissue turns brown, and how it colors the agar also.  I have not yet looked at these under a germicidal bulb (254 nm), only the black light, and the literature refers to using 254 nm.

- Mr.Caterpillar

*  I suppose it's obvious, but the derivation for "purpurea" is from the latin word for purple, "purpur", and presumably relates to the fungus's tendency towards purple.

**  On agar, purple excretion when seen through white mycelium has a definite violet cast to it, however I would not call the mycelium violet in this case.


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OfflineMr.Caterpillar
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Eywa_devotee]
    #19221636 - 12/03/13 11:21 AM (10 years, 2 months ago)

So, I gave this a shot, however I could not get very far owing to the physical difficulty of cutting the tiny and hard sclerotia in half.  I suppose with the right instruments one could do this, but I don't have those.

- Mr.Caterpillar


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Offlineshopdropper
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Mr.Caterpillar]
    #19221658 - 12/03/13 11:28 AM (10 years, 2 months ago)

i thought that "purpurea" was in reference to deep purple color of the sclerotia? well i know i read that somewhere recently. 

keep up the good work.


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DON'T TRY THIS AT HOME: no guarantees can be made about the accuracy of the information herein. The information dicussed in these posts is purely hypothetical, and for intelectual purposes only. Any similarity between internet chat and real life is pure coincidence.

:google:


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Offlineshopdropper
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19221672 - 12/03/13 11:30 AM (10 years, 2 months ago)

i think they used sand because it was easy to sterilize, held moisture pretty well and had no nutrition for competitors


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DON'T TRY THIS AT HOME: no guarantees can be made about the accuracy of the information herein. The information dicussed in these posts is purely hypothetical, and for intelectual purposes only. Any similarity between internet chat and real life is pure coincidence.

:google:


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InvisibleWillSolvem
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: shopdropper]
    #19226038 - 12/04/13 10:29 AM (10 years, 2 months ago)

:popcorn:


--------------------


AMU Q&A Thread because questions deserve answers.


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Offlinegoldenroad08
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: WillSolvem]
    #19234294 - 12/05/13 10:27 PM (10 years, 2 months ago)

subbed


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Trades


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: goldenroad08]
    #19234363 - 12/05/13 10:48 PM (10 years, 2 months ago)

:popcorn:


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Re: Starting Claviceps Paspali cultures from wild specimens [Re: Couperj]
    #20256005 - 07/10/14 11:49 PM (9 years, 7 months ago)

.


Edited by spore baby (12/13/14 02:30 PM)


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OfflineShroomDoom
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: spore baby]
    #20951673 - 12/09/14 09:53 AM (9 years, 2 months ago)

Quote:

spore baby said:
I wonder what happened to all of this?

Anyone know anything?



Yes I am eager to see what came of this.


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Offlinepiracetam
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #25455877 - 09/12/18 07:53 PM (5 years, 5 months ago)

sorry to necropost, but I found several specimens at a local park, and aseptically transferred some honeydew to PDA.
I'll have a go at it like the OP, and characterize.

I did a year and a half of work with an ATCC strain that fell in my lap, 7 years ago. Here are some pics of that work






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"Experiments are the only means of attaining knowledge at our disposal. The rest is just poetry, imagination." ~Max Planck


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Offlinepiracetam
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: piracetam]
    #25455881 - 09/12/18 07:55 PM (5 years, 5 months ago)




--------------------
"Experiments are the only means of attaining knowledge at our disposal. The rest is just poetry, imagination." ~Max Planck


Edited by piracetam (09/12/18 08:06 PM)


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OfflineShroomDoom
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: piracetam]
    #25486306 - 09/24/18 06:55 PM (5 years, 4 months ago)

:justastonishing:
outstanding work. Care to elucidate what some of those peaks are on Mass Spec?


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Offlinepiracetam
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Re: Starting Claviceps Paspali cultures from wild specimens [Re: ShroomDoom]
    #25486499 - 09/24/18 07:49 PM (5 years, 4 months ago)

thanks
pre-filtration extract, mostly clavines, such as chanoclavine II


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"Experiments are the only means of attaining knowledge at our disposal. The rest is just poetry, imagination." ~Max Planck


Edited by piracetam (09/24/18 07:59 PM)


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